Interplay between manganese and iron in pneumococcal pathogenesis: role of the orphan response regulator RitR

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that is carried asymptomatically in the nasopharynx by up to 70% of the human population. Translocation of the bacteria into internal sites can cause a range of diseases, such as pneumonia, otitis media, meningitis, and bacteremia. This transition from nasopharynx to growth at systemic sites means that the pneumococcus needs to adjust to a variety of environmental conditions, including transition metal ion availability. Although it is an important nutrient, iron potentiates oxidative stress, and it is established that in S. pneumoniae, expression of iron transport systems and proteins that protect against oxidative stress are regulated by an orphan response regulator, RitR. In this study, we investigated the effect of iron and manganese ion availability on the growth of a ritR mutant. Deletion of ritR led to impaired growth of bacteria in high-iron medium, but this phenotype could be suppressed with the addition of manganese. Measurement of metal ion accumulation indicated that manganese prevents iron accumulation. Furthermore, the addition of manganese also led to a reduction in the amount of hydrogen peroxide produced by bacterial cells. Studies of virulence in a murine model of infection indicated that RitR was not essential for pneumococcal survival and suggested that derepression of iron uptake systems may enhance the survival of pneumococci in some niches

Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393

A Novel Manganese Efflux System, YebN, Is Required for Virulence by Xanthomonas oryzae pv. oryzae

Manganese ions (Mn2+) play a crucial role in virulence and protection against oxidative stress in bacterial pathogens. Such pathogens appear to have evolved complex mechanisms for regulating Mn2+ uptake and efflux. Despite numerous studies on Mn2+ uptake, however, only one efflux system has been identified to date. Here, we report on a novel Mn2+ export system, YebN, in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight. Compared with wild-type PXO99, the yebN mutant was highly sensitive to Mn2+ and accumulated high concentrations of intracellular manganese. In addition, we found that expression of yebN was positively regulated by Mn2+ and the Mn2+-dependent transcription regulator, MntR. Interestingly, the yebN mutant was more tolerant to methyl viologen and H2O2 in low Mn2+ medium than PXO99, but more sensitive in high Mn2+ medium, implying that YebN plays an important role in Mn2+ homoeostasis and detoxification of reactive oxygen species (ROS). Notably, deletion of yebN rendered Xoo sensitive to hypo-osmotic shock, suggesting that YebN may protect against such stress. That mutation of yebN substantially reduced the Xoo growth rate and lesion formation in rice implies that YebN could be involved in Xoo fitness in host. Although YebN has two DUF204 domains, it lacks homology to any known metal transporter. Hence, this is the first report of a novel metal export system that plays essential roles in hypo-osmotic and oxidative stress, and virulence. Our results lay the foundations for elucidating the complex and fascinating relationship between metal homeostasis and host-pathogen interactions

The mechanism by which manganese protects Escherichia coli from hydrogen peroxide

The goal of this study was to understand the mechanism by which manganese protects cells against reactive oxygen species, particularly H2O2. We showed while manganese transport mutants, mntH, have no growth defect, mntH mutants in strains that cannot scavenge H2O2, Hpx- mntH, failed to grow when cultured aerobically. Thus manganese import is only crucial when cells are stressed with H2O2; cells appear to use iron to metallate mononuclear enzymes otherwise. Other workers have observed that manganese improves the ability of a variety of microbes to tolerate oxidative stress, and the prevailing hypothesis is that manganese does so by chemically scavenging hydrogen peroxide and/or superoxide .We found that manganese does not protect peroxide-stressed cells by scavenging peroxide. Instead, the beneficial effects of manganese correlate with its ability to metallate mononuclear enzymes. Because iron-loaded enzymes are vulnerable to the Fenton reaction, the substitution of manganese may prevent protein damage. Accordingly, during H2O2 stress, mutants that cannot import manganese and/or are unable to sequester iron suffer high rates of protein oxidation. To directly test our hypothesis, I studied three functionally distinct mononuclear enzymes: peptide deformylase (PDF), threonine dehydrogenase (TDH) and cytosine deaminase (CDA). We showed that these enzymes use iron as their cofactor, and that manganese functionally replaces iron and therefore protects these enzymes when cells are stressed with H2O2. We believe iron is frequently overlooked due to quick oxidation of ferrous iron to the insoluble ferric form in aerobic buffers. There are over two hundred mononuclear enzymes in E. coli, quite a few of which could be using iron in non-stressed conditions and thus during H2O2-stressed conditions could be using manganese. This implies that damage to iron-loaded enzymes is a global problem for cells. As we have demonstrated before, manganese import is critical for cell survival under H2O2 stress. In fact, the key metabolic failure of Hpx- mntH cells is due to lack of PDF activity: overexpressing PDF allows these cells to grow. We also demonstrated that these enzymes use manganese as their cofactor in iron-starved but H2O2-scavenging cells, which precludes the role of manganese as a scavenger. This conclusion is also supported by the ability of cobalt to relieve the cellular dependence on manganese during H2O2 stress. In this study, we also demonstrated that apo-PDF and apo-TDH are sensitive to H2O2. Both PDF and TDH have a metal-coordinating cysteine residue. While H2O2 is known to oxidize free cysteine, it does so at a maximal rate of 2 M-1 s-1 at neutral pH (half-life of inactivation at 1 μM H2O2 ~ 96 h), which is not fast enough to be physiologically relevant. In contrast, we have demonstrated that PDF and TDH are oxidized at a physiologically relevant rate of 1000-1300 M-1 s-1 (half-life of inactivation at 1 μM H2O2 ~ 10 min). These two enzymes are the first examples of proteins with cysteine residues that react with H2O2 at such a fast rate. We believe that H2O2-senstive active site cysteine residues in these enzymes are preferentially oxidized by H2O2 as a mechanism to spare other protein residues from irreversible covalent damage. This oxidized cysteine can then be later repaired by a reductant. Consistent with this idea, we have shown that oxidized inactivated proteins can be easily restored to full activity by a reductant in vitro. We have also only been able to retrieve proteins with cysteinyl residues in the over-oxidized state, which implies that the cell repairs oxidized proteins quite rapidly in vivo

Vergleich der Schmelzhaftung von Scotchbond Universal, Optibond Universal und Prime & Bond Active unter Verwendung der Total-Etch-Technik und Self-Etch-Technik

In den letzten Jahren stieg das Marktangebot an neuen Universal Bondingsystemen. Es wurde bisher noch wenig erforscht, ob diese neuen Produkte in vitro unter unterschiedlichen Applikationsbedingungen in Bezug auf ihre Schmelzhaftung gleich gut abschneiden. Die Produkte Scotchbond Universal, OptiBond Universal und Prime&Bond active® wurden unter Self-Etch und Total-Etch- Technik auf ihre Haftwirkung am Schmelz getestet. Material und Methodik Für die Versuchsreihe wurden 240 bovine Zähne verwendet. Dabei wurden sie in drei Gruppen zu je 80 Zähne unterteilt, jeweils die Hälfte davon für die zwei verschiedenen Applikationsformen (Total-Etch/Self-Etch). Am Schmelz wurde eine plane Oberfläche beschliffen. Auf der Schmelzoberfläche wurden die Adhäsivsysteme laut Herstellerangaben appliziert. Anschließend wurde darauf ein Kompositprüfkörper aus CeramX-Universal mit einem Durchmesser von 4mm im rechten Winkel appliziert. Die Prüfkörper wurden 24 Stunden bei 37C in Wasser gelagert. Bei den Scherversuchen wurden die Proben in Gips eingebettet und die Kompositzylinder anschließend mittels Universal Test Maschine (Zwick) mit einer Vorschubgeschwindigkeit von 0,8mm/min abgeschert. Die Haftwerte sowie das Bruchverhalten der Proben wurden ermittelt. Ergebnisse Die Total-Etch Applikation von Scotchbond Universal zeigte im Vergleich zu OptiBond Universal signifikant höhere Haftwerte. Die Ergebnisse für die Self-Etch Technik zeigten bei der Verwendung von Prime&Bond active® im Vergleich zu OptiBond Universal und auch im Vergleich zu Scotchbond Universal signifikant niedrigere Haftwerte. Bei Scotchbond Universal und Prime&Bond active® wurden bei Verwendung der Total-Etch Anwendung im Vergleich zur Self-Etch Technik signifikant höhere Haftwerte festgestellt. Nur bei OptiBond Universal wurden unabhängig von der Applikationstechnik ähnliche Haftwerte festgestellt. Kohäsive Brüche wurden hauptsächlich bei der Total-Etch-Technik beobachtet. Konklusion Die signifikant höhere Haftfestigkeit auf Schmelz bei der Anwendung der Total-Etch Technik von Scotchbond Universal und Prime&Bond active® könnten auf die Überlegenheit der Phosphorsäurevorkonditionierung im Vergleich zur intrinsischen Vorkonditionierung hinweisen. Nur bei OptiBond Universal erzielte die intrinische Vorkonditionierung des Schmelzes ähnlich hohe Haftwerte wie bei der Vorkonditionierung durch Phosphorsäure.In the last years many different single-step adhesives were introduced into the market. There is still a lack of evidence if these newer formulations perform equally whether used in the self-etch technique or in the total-etch technique. Against this background until now a relatively small number of publications analyze the shear bond strength of single-step adhesives to enamel. Material and Methods The products Scotchbond Universal, OptiBond Universal and Prime&Bond active® were investigated with special focus on differences of shear bond strength values to enamel comparing the self-etch technique with the total-etch technique. 240 bovine teeth were tested, divided in three groups of 80 for each product; half of them were preconditioned with phosphoric acid. Furthermore, the enamel was prepared, to obtain a plain 5mm diameter surface. The products were applied following the instructions of the manufacturers. Afterwards a composite specimen of CeramX-Universal with a diameter of 4 mm was applied at a right angle. Test specimens were stored for 24 hours in water at 37C. Before the tests the specimens were stabilized in plaster. The bond strength between the composite specimen and the enamel was determined by the universal test machine (Zwick) with a cross head speed of 0,8mm/min. Bonding strength values and the fracture behavior were analyzed. Results The total-etch application of Scotchbond Universal showed significant higher bond strengths compared to OptiBond Universal. Results for the self-etch technique exhibited significant lower bond strengths using Prime&Bond active®, compared to OptiBond Universal and to Scotchbond Universal. Significant higher bond strengths were found for Scotchbond Universal and Prime&Bond active® when using the total-etch application compared to the self-etch technique. Only for OptiBond Universal a similar bonding strength was found regardless of application technique. Cohesive failures were mainly investigated when using the total-etch technique. Conclusion The significant higher bond strength on enamel using the total-etch technique for Scotchbond Universal and Prime&Bond active® may indicate the superiority of the phosphoric acid preconditioning compared to the intrinsic preconditioning of the adhesive system. OptiBond Universal directly applied, could precondition the enamel similar to the total-etch protocol with phosphoric acid.Paralleltitel laut Übersetzung des VerfassersMedizinische Universität Wien, Diplomarbeit, 2019(VLID)456846

Påvirkningen av business analytics modenhet på individers makt i bedrifter

en verden med store mengder data tilgjengelig fokuserer bedriftene mer på å utvikle sine business analytics kapabiliteter for å overleve i det tøffe konkurransemarkedet og øke lønnsomheten sin (Takeuchi, 2016). Det gir tilgang til ny informasjon og verdifull innsikt som kan ha en effekt på individers makt i bedrifter (Astley & Sachdeva, 1984). Noen individer i organisasjonen kan få lettere tilgang til informasjon og bruke den til å påvirke andre, mens andre kan miste sin makt fordi de ikke lenger er like viktige for å skaffe verdifull innsikt (Lee, 1991). Denne oppgaven studerer innvirkningen business analytics modenhet har på individuell makt i bedrifter, og om denne sammenhengen påvirkes av ferdighetene individene besitter. For å skille mellom ferdighetene er det tatt utgangspunkt i myke og harde ferdigheter. Forskningsmodellen som er utviklet tar utgangspunkt i eksisterende teori innenfor makt, business analytics og ferdigheter. I teorigrunnlaget utdypes maktkilder som blant annet autoritet, ressurskontroll, nettverkssentralitet, ikke-substituerbarhet og ekspertise. Videre redegjøres det for modenhetsmodeller innenfor business analytics, før myke og harde ferdigheter belyses. For å undersøke den konseptuelle modellen er det brukt en kvantitativ tilnærming. Det er benyttet nettbasert spørreskjema som datainnsamlingsmetode og det er samlet inn data fra 66 individer som er en del av arbeidslivet i Norge i en tverrsnittsundersøkelse. Den innsamlede dataen er analysert ved bruk av programvaren SPSS. Resultatene viser som forventet at det eksisterer en sammenheng som tilsier at økt business analytics modenhet bidrar til mer makt. Videre viser funnene at ulike nivåer av harde ferdigheter moderer denne relasjonen, denne studien lykkes ikke med å påvise en tilsvarende sammenheng for myke ferdigheter. Funnene fører frem til at individer opplever økt maktbesittelse som følge av at bedriften de jobber blir mer moden innenfor business analytics, og at individer bør være bevisste på sine harde ferdigheter i denne relasjonen og utvikle disse til et moderat nivå for å oppnå høyere makt i forhold til individer med harde ferdigheter på et høyt eller lavt nivå

New insights into the protective effect of manganese against oxidative stress

Manganese has emerged as an important trace element in bacterial physiology. The correlation between manganese accumulation and resistance to oxidative stress has led to the suggestion that, in addition to a role as a prosthetic group in superoxide dismutase, manganese could exert its antioxidant effect via non-enzymatic redox reactions. The article by Anjem et al. in the current issue of Molecular Microbiology investigates the role of manganese ions in the defence against hydrogen peroxide in Escherichia coli. The results indicate that the redox activity of manganese is not linked to its protective effect. Instead, it is suggested that manganese replaces ferrous iron in enzymes that contain divalent cations at their active site. This enables the cell to avoid oxidative stress associated with iron release in the presence of hydrogen peroxide

Protein damage and death by radiation in Escherichia coli and Deinococcus radiodurans

Deinococcus radiodurans is among a small number of bacterial species that are extremely resistant to ionizing radiation, UV light, toxic chemicals, and desiccation. We measured proteome oxidation (i.e., protein carbonylation, PC) in D. radiodurans as well as in standard and evolved resistant strains of Escherichia coli exposed to ionizing radiation or UVC light and found a consistent correlation with cell killing. The unique quantitative relationship between incurred PC and cell death holds over the entire range of killing for all tested bacteria and for both lethal agents, meaning that both bacterial species are equally sensitive to PC. We show that the extraordinary robustness of D. radiodurans depends on efficient proteome protection (but not DNA protection) against constitutive and radiation-induced PC consisting of low molecular weight cytosolic compounds. Remarkably, experimental evolution of resistance to ionizing radiation in E. coli coevolves with protection against PC. The decline in biosynthetic efficacy of the cellular proteome, as measured by the loss of reproduction of undamaged bacteriophage λ in irradiated standard and evolved ionizing radiation-resistant E. coli, correlates with radiation-induced oxidative damage to host cells and their sensitivity to ionizing radiation. This correlation suggests that cell death by radiation is caused primarily by oxidative damage with consequential loss of maintenance activities including DNA repair